Editing Prepare-Cel1-from-Celery

== '''Make Cel-1 Nuclease&nbsp; from Celery'''  ==

== Protocol 1  ==

Note:<br>â¢ Have ready all PMSF and Tris-Buffer solutions<br>â¢ Thaw aliquots of PMSF solution immediately before use<br>â¢ Use a sieve to remove floating debris<br><br>

1.&nbsp; Prepare celery for juicing:<br>&nbsp;&nbsp;&nbsp; a. Cut off the bottom from each bunch to separate stalks<br>&nbsp;&nbsp;&nbsp; b. Trim off tops, leaves, and tough, white bottoms from each stalk<br>&nbsp;&nbsp;&nbsp; c. Rinse with water and dry with paper towels<br>2.&nbsp; Blender celery at 4° C to obtain 1.5 L juice<br>3.&nbsp; Make a 0.1 M Tris-HCL, pH 7.7, 100 ÎŒM PMSF solution in celery juice<br>&nbsp;&nbsp;&nbsp;&nbsp; a. per L juice add: 50 ml 2M Tris pH 7.7<br>&nbsp;&nbsp;&nbsp;&nbsp; b. per L juice add: 1 ml 0.1 M PMSF<br>4.&nbsp; Centrifuge the juice solution at 15,000 g for 20 minutes, 4 °C<br>5.&nbsp; Decant and save supernatant, discard pellet<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: fibrous and sticky, bright green<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: cloudy greenish-yellow<br>6.&nbsp; Bring supernatant to 25% ammonium sulfate<br>&nbsp;&nbsp;&nbsp;&nbsp; a. per L supernatant add: 144 g (NH4)2SO4<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Volume will increase marginally (3%)<br>7.&nbsp; Mix solution gently for 1 hour, 4 °C<br>8.&nbsp; Centrifuge solution at 15,000 g for 45 minutes, 4 °C<br>9.&nbsp; Decant and save supernatant, discard pellet<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: small and sticky, green or light gray<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: cloudy brownish-yellow<br>10.&nbsp; Bring supernatant to 80% ammonium sulfate<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Per L supternatant add: 390 g (NH4)2SO4<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Volume will increase significantly (25%)<br>11.&nbsp; Mix solution gently for 1 hour or overnight, 4 °C<br>12.&nbsp; Centrifuge solution at 15,000 g for 90 minutes, 4 °C<br>13.&nbsp; Save pellet, decant and discard supernatant<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: large compared to previous pellets, yellowish-brown or beige<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: transparent brownish-yellow<br>14.&nbsp; Store protein-precipitate pellet at 4 °C (weeks) or -80 °C (indefinite) 

=== Dialysis<br>  ===

'''Caution:'''<br>â¢ Use Gloves in All Steps, Note that PMSF is TOXIC.<br>â¢ PMSF will recrystallize at 4 °C â thaw and use immediately<br>â¢ PMSF will degrade over time in aqueous solutions â do not allow to sit in solution<br>

'''Prepare 5 L Buffer B''':&nbsp; <br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 374px; height: 146px;"
|-
| a. 0.1 M Tris pH 7.7<br>
| add 50 ml 2 M stock
|-
| b. 0.5 M KCl
| add 250 ml 2 M stock
|-
| c. 0.01% Triton X-100 
| add 5 ml 20% stock
|-
| d. 100 ÎŒM PMSF
| add 5 ml 0.1 M stock
|}

'''Resuspend pellets in a volume of Buffer B equivalent to 1/10th the initial volume of celery juice'''<br>&nbsp;&nbsp; a. For a 1.5 L prep, resuspend in a 150 ml solution of protein in Buffer B<br>&nbsp;&nbsp; b. Scrape pellet from wall of centrifuge bottle using a metal spatula<br>&nbsp;&nbsp; c. Smear or break down large chunks of pellets for easier solvation<br>&nbsp;&nbsp; d. Vortex gently by hand and avoid over-foaming solution<br>&nbsp;&nbsp; e. Transfer buffer and pellets from tube to tube until all pellets are combined<br>&nbsp;&nbsp; f. Resuspended pellet should be homogeneous and free of debris<br> 

'''Add suspended protein to dialysis tubes'''<br>

'''Dialyze in 5 or 6 changes of 5 L Buffer B'''<br>&nbsp;&nbsp; a. Stir solution slowly to avoid drawing dialysis tubes down into the stir bar<br>&nbsp;&nbsp; b. Allow last dialysis to proceed for at least 4 hours or overnight<br>

'''Collect the dialyzed protein solution and aliquot'''<br>&nbsp;&nbsp; Aliquot into labeled and dated 1.5 or 2.0 ml tubes<br>

'''Store aliquots at -80 °C'''<br>



== '''Protocol 2''' ==

1. Add 500 ml of 0.1 M TrisHCl, pH 7.7 with 100 µM PMSF to 0.5 kg of store-bought celery.<br>2. Chop with a blender at 4°C.<br>3. Spin for 20 min at 6000 rpm to pellet debris.<br>4. Add (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> to supernatant to adjust to 25% saturation.<br>5. Put on ice for 30 min.<br>6. Spin at 12000 rpm for 30 min at 4°C <br>7. Add (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> to the resulting supernatant to 80% saturation.<br>8. Put on ice for 30 min.<br>9. Spin at 25000 rpm for 30 min. <br>10. Resuspend the pellet in 50 ml of 0.1 M Tris-HCl, pH 7.7, 100 mM PMSF (1/10 starting volume). <br>11. Dialyse the suspension with 10K MW cut-off against a total of 8L of the same buffer, with four changes every 4 h.<br>12. Store aliquots of extract at -20°C or -80°C 

== '''Protocol 3'''<br>  ==

1. Use a Blender to chop Celery stalks (7 kg) at 4 <sup>o</sup>C.<br>2. Adjusted with a 10Ã solution to give the composition of buffer A (0.1 M TrisâHCl, pH 7.7, 10 mM phenylmethanesulfonyl<br>fluoride (PMSF).<br>3. Spin for 20 min at 6000 rpm to pellet debris.<br>4. Add (NH4)2SO4 to supernatant to adjust to 20% saturation.<br>5. Put on ice for 30 min.<br>6. Spin at 12000 rpm for 30 min at 4°C<br>7. Add (NH4)2SO4 to the resulting supernatant to 70% saturation.<br>8. Put on ice for 30 min.<br>9. Spin at 25000 rpm for 30 min. 

10. Dissolve the final pellet in 250 ml buffer A and dialyze against 0.5 M KCl in buffer A. 

11. Incubate the solution with 10 ml concanavalin AâSepharose resin (Sigma) overnight at 4 oC. 

12. Pack the slurry into a 2.5 cm diameter column and wash with 0.5 M KCl in buffer A. 

13. Elute bound CEL-I with 90 ml 0.3 M a-D+-mannose, 0.5 M KCl in buffer A at 65 oC. 

14. Dialyse CEL-I against buffer B (25 mM potassium phosphate, 10 mM PMSF, pH 7.0). 

15. Apply the dialysed solution to a 100 ml phosphocellulose P-11 column that had been equilibrated in buffer B. 

16. Elute the bound enzyme with a linear gradient of KCl in buffer B. 

17. Pool fractions containing&nbsp; CEL-I activity. 

18. Add ammonium sulfate to saturation, incubate on ice for 30 min. 

19. Spin to collect precipitate. 

20. Dissolve the precipitate in Buffer C (50 mM TrisâHCl, pH 7.8, 0.2 M KCl, 10 mM PMSF, 1 mM ZnCl<sub>2</sub>). 

21. Dialyse against Buffer C. 

22. Load to a Superose 12 FPLC column equilibrated with Buffer C.<br> 

== '''Digestion Buffer and Condition'''  ==

After PCR gene synthesis, to 50 µL PCR reaction add:<br> 

10 µL of '''10X digestion buffer '''(100 mM KCl, 100 mM MgSO<sub>4</sub>, 100 mM Tris-HCl, pH 7.5, 0.02% Triton X100, 0.002 mg/ml BSA)<br> 

5 µL of Cel-1 Nuclease<br> 

35 µL water<br> 

Incubate at 45°C for 15 min<br> 

<br> 

Alternative Condition 

CEL 1 digestion solution: 10mM HEPES (pH 7.5), 10 mM MgS0<sub>4</sub>, 0.002% (w/v) Triton X-100, 20 ng/ml bovine serum albumin, and 1:300-1:10,000 dilution of CEL 1.<br> 

[[Category:Protocols]]